Cellular Reservoirs Research Articles

Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca2+, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca2+ dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca2+. Here we describe a robust and quantitative method based on the Ca2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca2+ such that it conforms with the [Ca2+] values found in the ER and it slows the consumption of the probe by Ca2+. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1h) ER Ca2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca2+]ER and Ca2+ dynamics in intact cells stimulated with IP3-produced agonists. In addition, GAP1 can record Ca2+ mobilization in permeabilized cells challenged with IP3. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca2+]ER.

Primordial Germ Cells Quality of Chicken Embryo from Post Cryopreservation Blood Samples

The optimal cellular reservoir for the conservation and propagation of endangered poultry is acknowledged to be primordial germ cells (PGCs). This study aimed to evaluate and compare the efficacy of cryopreserving PGCs derived from purified sources and blood samples. The experimental design encompassed two treatment groups, each iterated six replication. Blood samples that had been filled with 500 µL of phosphate-buffered saline without Ca2+ and Mg2+ (PBS) underwent centrifugation at 1200 rpm for six minutes. The supernatant was discarded and the pellet was mixed with 500 µL 10% FBS-PBS and was then subdivided for cryopreservation and PGCs purification. Cryopreservation included the controlled freezing of blood and purified PGCs samples in Nalgene cryogenic vials with the incorporation of banker cells. Following thawing, statistical analyses revealed no significant variance in the mean PGCs count between purified PGCs and blood samples (p > 0.05). The percentages of PGCs mortality and the recovery rate demonstrated statistical significance (p < 0.05). Further investigations indicated that cryopreserving blood samples significantly increased the recovery rate of PGCs while decreasing post-thaw mortality. Morphology of PGCs from blood cryopreservation showed similar morphology to PGCs in general. The potential application of cell-based cryo-banking will contribute to the preservation of poultry genetic diversity for sustainability and adaptation to future poultry demands.

New Therapies and Strategies to Curb HIV Infections with a Focus on Macrophages and Reservoirs.

More than 80 million people worldwide have been infected with the human immunodeficiency virus (HIV). There are now approximately 39 million individuals living with HIV/acquired immunodeficiency syndrome (AIDS). Although treatments against HIV infection are available, AIDS remains a serious disease. Combination antiretroviral therapy (cART), also known as highly active antiretroviral therapy (HAART), consists of treatment with a combination of several antiretroviral drugs that block multiple stages in the virus replication cycle. However, the increasing usage of cART is inevitably associated with the emergence of HIV drug resistance. In addition, the development of persistent cellular reservoirs of latent HIV is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Thus, several efforts are being applied to new generations of drugs, vaccines and new types of cART. In this review, we summarize the antiviral therapies used for the treatment of HIV/AIDS, both as individual agents and as combination therapies, and highlight the role of both macrophages and HIV cellular reservoirs and the most recent clinical studies related to this disease.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

Optimization of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.

Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation.

Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.

Potential role of alveolar macrophages in HIV persistence and lung disease

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T-cells, and particularly memory CD4+ T-cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory co-infections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

Potential role of alveolar macrophages in HIV persistence and lung disease.

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T cells, and particularly memory CD4+ T cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory coinfections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

Mitotic deacetylase complex (MiDAC) recognizes the HIV-1 core promoter to control activated viral gene expression.

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.

Low-level viremia episodes appear to affect the provirus composition of the circulating cellular HIV reservoir during antiretroviral therapy.

Low-level viremia (LLV) ranging from 50 to 1,000 copies/ml is common in most HIV-1-infected patients receiving antiretroviral therapy (ART). However, the source of LLV and the impact of LLV on the HIV-1 reservoir during ART remain uncertain. We hypothesized that LLV may arise from the HIV reservoir and its occurrence affect the composition of the reservoir after LLV episodes. Accordingly, we investigated the genetic linkage of sequences obtained from plasma at LLV and pre-ART time points and from peripheral blood mononuclear cells (PBMCs) at pre-ART, pre-LLV, LLV, and post-LLV time points. We found that LLV sequences were populated with a predominant viral quasispecies that accounted for 67.29%∼100% of all sequences. Two episodes of LLV in subject 1, spaced 6 months apart, appeared to have originated from the stochastic reactivation of latently HIV-1-infected cells. Moreover, 3.77% of pre-ART plasma sequences were identical to 67.29% of LLV-3 plasma sequences in subject 1, suggesting that LLV may have arisen from a subset of cells that were infected before ART was initiated. No direct evidence of sequence linkage was found between LLV viruses and circulating cellular reservoirs in all subjects. The reservoir size, diversity, and divergence of the PBMC DNA did not differ significantly between the pre- and post-LLV sampling points (P > 0.05), but the composition of viral reservoir quasispecies shifted markedly before and after LLV episodes. Indeed, subjects with LLV had a higher total PBMC DNA level, greater viral diversity, a lower proportion of variants with identical sequences detected at two or more time points, and a shorter variant duration during ART compared with subjects without LLV. Overall, our findings suggested that LLV viruses may stem from an unidentified source other than circulating cellular reservoirs. LLV episodes may introduce great complexity into the HIV reservoir, which brings challenges to the development of treatment strategies.

Characterization of Neisseria gonorrhoeae colonization of macrophages under distinct polarization states and nutrients environment.

Neisseria gonorrhoeae (Ng) is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. Ng has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. Ng successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, Ng manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by Ng. Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that Ng can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.

HIV-1 Myeloid Reservoirs - Contributors to Viral Persistence and Pathogenesis.

HIV reservoirs are the main barrier to cure. CD4+ T cells have been extensively studied as the primary HIV-1 reservoir. However, there is substantial evidence that HIV-1-infected myeloid cells (monocytes/macrophages) also contribute to viral persistence and pathogenesis. Recent studies in animal models and people with HIV-1 demonstrate that myeloid cells are cellular reservoirs of HIV-1. HIV-1 genomes and viral RNA have been reported in circulating monocytes and tissue-resident macrophages from the brain, urethra, gut, liver, and spleen. Importantly, viral outgrowth assays have quantified persistent infectious virus from monocyte-derived macrophages and tissue-resident macrophages. The myeloid cell compartment represents an important target of HIV-1 infection. While myeloid reservoirs may be more difficult to measure than CD4+ T cell reservoirs, they are long-lived, contribute to viral persistence, and, unless specifically targeted, will prevent an HIV-1 cure.

Oxidative stress, microparticles, and E-selectin do not depend on HIV suppression.

Oxidative stress and inflammation are considered predictors of diseases associated with aging. Markers of oxidative stress, inflammation, and endothelial activation were investigated in people with HIV on antiretroviral treatment to determine whether they had an immunosenescent phenotype that might predispose to the development of premature age-related diseases. This study was conducted on 213 subjects with HIV. The control groups consisted of healthy HIV-negative adults. The level of oxidative stress was measured by assessing the production of malondialdehyde levels, which were detected by thiobarbituric acid reactive substance (TBARS) assay. The level of microparticles indicated the presence of inflammation and endothelial activation was measured by E-selectin levels. Significant differences were determined by appropriate statistical tests, depending on the distribution of variables. Relationships between continuous variables were quantified using Spearman's rank correlation coefficient. TBARS, and microparticle and E-selectin levels were significantly higher in untreated and treated subjects with HIV compared with HIV-negative controls (P<0.001). The levels of the investigated markers were not significantly different between untreated and treated patients and no significant correlation of these markers was found with CD4+ count, CD4+/CD8+ ratio, and the number of HIV-1 RNA copies. Elevated markers of oxidative stress, inflammatory and endothelial activation were independent of the virologic and immunologic status of people with HIV. These results support the hypothesis that residual viremia in cellular reservoirs of various tissues is a key factor related to the premature aging of the immune system and predisposition to the premature development of diseases associated with aging.

Formulation and Evaluation of Lipid Based Nanoparticles of Etravirine

Engineered nanoparticles have the potential to revolutionize the diagnosis and treatment of many diseases like HIV/AIDS. Etravirine is one of the key components of highly active antiretroviral therapy used for the treatment of HIV-1 infections. The aim of the present study was to formulate and evaluate nanostructured lipid carriers of etravirine, intended for targeted delivery to macrophages, using solvent emulsification - evaporation technique. Estimates of drug solubility were employed for selection of solid lipids, liquid lipids and stabilizers for the preparation of NLCs. Design of experiments was used to optimize the formulation with respect to drug-lipid ratio and concentration of stabilizer in the external phase using 32 full factorial design. Particle size of the carriers and drug release characteristics were the responses which were set to suitable levels for optimization. The optimized formulation was prepared and characterized for size, poly dispersity index, zeta potential, entrapment efficiency and appearance. The nanostructured lipid carriers of etravirine were prepared using stearylamine and glyceryl monostearate as solid lipids, Capryol 90 as liquid lipid and polyvinyl pyrrolidone as stabilizer. All experimental batches showed high drug loading efficiencies nearing 99%, indicating that etravirine remained closely associated with the lipids. The nanostructured lipid carriers displayed a zeta potential of -10.1 mv and a particle size of 261.6 nm with a polydispersity index of 0.374. In vitro release of etravirine from the optimized formulation at 2 h was 9% indicative of a low burst; and 56% of the entrapped drug was released after 24 h, suggesting prolonged release characteristics. Thus, etravirine loaded lipidic nanoparticles with potential for targeting cellular reservoirs of the AIDS virus such as macrophages were successfully developed.&#x0D; Keywords: Etravirine, Nanostructured lipid carriers, full factorial design

Biocompatible metal-organic frameworks as promising platforms to eradicate HIV reservoirs ex vivo in people living with HIV.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

The latent HIV reservoir: current advances in genetic sequencing approaches.

Multiple cellular HIV reservoirs in diverse anatomical sites can undergo clonal expansion and persist for years despite suppressive antiretroviral therapy, posing a major barrier toward an HIV cure. Commonly adopted assays to assess HIV reservoir size mainly consist of PCR-based measures of cell-associated total proviral DNA, intact proviruses and transcriptionally competent provirus (viral RNA), flow cytometry and microscopy-based methods to measure translationally competent provirus (viral protein), and quantitative viral outgrowth assay, the gold standard to measure replication-competent provirus; yet no assay alone can provide a comprehensive view of the total HIV reservoir or its dynamics. Furthermore, the detection of extant provirus by these measures does not preclude defects affecting replication competence. An accurate measure of the latent reservoir is essential for evaluating the efficacy of HIV cure strategies. Recent approaches have been developed, which generate proviral sequence data to create a more detailed profile of the latent reservoir. These sequencing approaches are valuable tools to understand the complex multicellular processes in a diverse range of tissues and cell types and have provided insights into the mechanisms of HIV establishment and persistence. These advancements over previous sequencing methods have allowed multiplexing and new assays have emerged, which can document transcriptional activity, chromosome accessibility, and in-depth cellular phenotypes harboring latent HIV, enabling the characterization of rare infected cells across restrictive sites such as the brain. In this manuscript, we provide a review of HIV sequencing-based assays adopted to address challenges in quantifying and characterizing the latent HIV reservoir.

Polarity-Driven Two-Photon Fluorescent Probe for Monitoring the Perturbation in Lipid Droplet Levels during Mitochondrial Dysfunction and Acute Pancreatitis.

Lipid droplets (LDs) act as an energy reservoir in cancer cells; on the other hand, mitochondria are hyperactive to fulfill the energy demand to accelerate cell proliferation. We are interested in unfolding the relationship between the cellular energy reservoir and energy producer through fluorescence labeling. Thus, a dual organelle-targeted fluorescent probe MLD-1 has been rationally developed. It visualized the crosstalk between mitochondrial dysfunction and the fluctuation of LDs in live cells. Its two-photon ability allowed us to acquire deep tissue images. For the first time, we have shown that the probe has the ability to track the accumulation of LDs in different mouse organs during pancreatic inflammation. MLD-1, being a selectively polarity-driven, chemo- and photostable LD probe, may offer great possibilities for studying LD-associated biology in due course.

Nature and evolution of the cellular HIV-1 reservoir in children and adolescents.

Shortly after primary infection, HIV hides in cellular reservoirs from which it becomes difficult or almost impossible to dislodge. In the absence of effective antiretroviral therapy, there is almost invariably resurgence of productive infection leading to a decline in CD4+ T cell counts and progression of HIV disease. The course of HIV infection in adults (horizontal transmission) differs significantly from that acquired in children following perinatal transmission: steady-state viral load is higher in children, adherence issues make it more difficult to control viral load using antiretroviral therapy, and the life expectancy of HIV-infected children in absence of treatment is markedly shorter than that of adults. Compared to the situation in adults, we know very little about the nature of the cellular reservoir in children, about its importance at the quantitative level, about its persistence over time, about its evolution during infancy, childhood and adolescence, and about its influence on the pathogenesis of pediatric HIV-AIDS. Some reported cases of spontaneous remission of HIV infection in children in the absence of treatment have also fueled the hopes of discovering avenues leading to a functional cure for HIV-AIDS in both children and adults.

Nature and evolution of the cellular HIV-1 reservoir in children and adolescents

Shortly after primary infection, HIV hides in cellular reservoirs from which it becomes difficult or almost impossible to dislodge. In the absence of effective antiretroviral therapy, there is almost invariably resurgence of productive infection leading to a decline in CD4+ T cell counts and progression of HIV disease. The course of HIV infection in adults (horizontal transmission) differs significantly from that acquired in children following perinatal transmission: steady-state viral load is higher in children, adherence issues make it more difficult to control viral load using antiretroviral therapy, and the life expectancy of HIV-infected children in absence of treatment is markedly shorter than that of adults. Compared to the situation in adults, we know very little about the nature of the cellular reservoir in children, about its importance at the quantitative level, about its persistence over time, about its evolution during infancy, childhood and adolescence, and about its influence on the pathogenesis of pediatric HIV-AIDS. Some reported cases of spontaneous remission of HIV infection in children in the absence of treatment have also fueled the hopes of discovering avenues leading to a functional cure for HIV-AIDS in both children and adults.